CN104988246A - Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method - Google Patents
Eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method Download PDFInfo
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Abstract
The invention relates to the technical field of breeding, in particular to an eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method. The eriocheir sinensis white spot syndrome virus specificity PCR detection kit comprises 1.0 mL of a 2*reaction mixture buffer solution, preferably-designed upstream or downstream primers, Taq DNA polymerase, positive control fluid, negative control fluid and ddH2O. The novel and rapid PCR detection kit and method overcome the defects of an existing eriocheir sinensis white spot syndrome virus detection method. The eriocheir sinensis white spot syndrome virus specificity PCR detection kit and method are practicable for clinic diagnosis on the eriocheir sinensis white spot syndrome virus, have the advantages of being rapid, accurate and special, meet the requirement of clinic diagnosis, and provide convenience for eriocheir sinensis white spot syndrome virus detection.
Description
Technical field
The present invention relates to cultural technique field, be specifically related to cause mitten crab morbidity cause of disease Leucodermia virus specific PCR quick detection kit and detection method, in particular to a kind of gene detection reagent and detection method of mitten crab morbidity cause of disease Leucodermia virus, be applicable to the fields such as the epidemiological surveillance of mitten crab Leucodermia virus, the detection of Leucodermia virus and genetic engineering technique.
Background technology
Leucodermia virus (WSSV) is the virus of the current shrimp culture industry of a kind of serious harm, mainly causes hickie pathology inside prawn crust, and causes large quantities of death.Its Epidemic Scope is wide, in crustaceans, copepods and insects, all have responsive host.
Mitten crab, also known as river crab, is a kind of important economic crab, and the Eriocheir sinensis industry development of China is rapid, established practice modelling.Aborning, in order to improve culture benefit, generally take the aquaculture model that multi items is raised together with, as Procambius clarkii and mitten crab are raised together with.But in recent years, after often there is Procambius clarkii death in enormous quantities, there is the phenomenon of mortality in the mitten crab be in identical culture pond simultaneously.Xue Hui etc. carry out Sampling Survey the Procambius clarkiis to the morbidity of some areas, Jiangsu cultivating pool in 2010 and mitten crab, confirm that the cause of disease causing these two kinds of fresh water Crustacean morbidities dead is Leucodermia virus.
Diagnostic means at present for Leucodermia virus disease mainly comprises pathology method, biological method, immunological method, molecular hybridization method and PCR method etc.But yet there are no the method fast and accurately detecting mitten crab, in order to find whether cultivation mitten crab is infected by Leucodermia virus, is necessary to set up a kind of quick, accurate, sensitive detection method early.PCR method has the advantages such as simple to operate, special, quick, responsive, is just progressively applied to the detection of pathogenic micro-organism with research.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, provides a kind of mitten crab Leucodermia virus PCR method for detecting specificity, can fast and efficiently for the detection of mitten crab Leucodermia virus.
The present invention is achieved through the following technical solutions.
The PCR quick detection kit of mitten crab Leucodermia virus, described test kit comprises:
(1) 2 × reaction cocktail buffer, comprises following composition: KCl, MgCl
2, Tris-HCl pH5.8, dNTP;
(2) design and synthesis of primer is detected: according to the conserved sequence of the Leucodermia virus gene issued in GenBank, design a pair specific detection primer, upstream primer WSSV-F:5 '-GTGTACTAGGAATATTGGAAT-3 ', downstream primer WSSV-R:5 '-CGGCATTCTTCATGGCTTCTG-3 ';
(3) Taq enzyme;
(4) ddH of sterilizing
2o;
(5) positive control solution: Leucodermia virus genomic dna;
(6) negative controls: ddH
2o.
The method adopting test kit to detect mitten crab Leucodermia virus is:
(1) extraction of mitten crab Leucodermia virus DNA: the gill getting mitten crab, hepatopancreas, muscle tissue, add the distilled water of sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 10 minutes, get supernatant, add the saturated phenol of Tris-, after abundant vibration mixing, leave standstill 5 minutes, then centrifugal 5 minutes of 12000rpm, get the phenol that supernatant liquor adds equivalent: chloroform: isoamyl alcohol extraction secondary, get the Virahol that supernatant adds 2 times of volumes, after mixing, leave standstill 10 minutes, centrifugal 10 minutes of 12000rpm, after removing supernatant, add the ethanol of 75%, leave standstill 5 minutes, centrifugal 10 minutes of 12000rpm, remove supernatant, after being placed in vacuum drying oven drying, use ddH
2o dissolves, and be test DNA profiling, the DNA profiling of extraction is placed in-70 DEG C and saves backup,
(2) PCR reaction system: adopt PCR reaction system, 2 × reaction cocktail buffer is added respectively in PCR reaction tubes, upstream primer, downstream primer, 5U/ μ L Taq archaeal dna polymerase, DNA profiling, adds sterilizing ultrapure water, use positive control solution and negative controls as template respectively simultaneously, the positive and negative control group are set, mix the rear centrifugal several seconds, be placed in PCR and react on instrument;
(4) PCR reaction conditions: 95 DEG C of denaturations 5 minutes; Then 95 DEG C of sex change 30 seconds, 56 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, circulate 30 times altogether; 72 DEG C extend 10 minutes, last 4 DEG C of preservations;
(5) after the detection of pcr amplification product: PCR reaction terminates, get product on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR primer, check the expection size of PCR primer;
(6) result and judgement: observe under ultraviolet lamp and contrast with standard molecular weight, if detect sample to occur bright band at 441bp place, and negative control does not have object band, be then that Leucodermia virus is positive; If the visible object band of positive control, and detect sample without object band and occur, then be feminine gender, do not have or be not the object Leucodermia virus that will detect.
Preferred test kit comprises:
(3) 2 × reaction cocktail buffer 1.0mL, comprises following composition:
(4) design and synthesis of primer is detected: according to the conserved sequence of the Leucodermia virus gene issued in GenBank, design a pair specific detection primer, upstream primer WSSV-F:5 '-GTGTACTAGGAATATTGGAAT-3 ', downstream primer WSSV-R:5 '-CGGCATTCTTCATGGCTTCTG-3 ', concentration is 10 μMs;
(3) Taq enzyme 5U/ μ L;
(4) ddH of sterilizing
2o 1.0mL;
(5) positive control solution: Leucodermia virus genomic dna;
(6) negative controls: ddH
2o.
The method that preferred employing test kit detects mitten crab Leucodermia virus is:
(1) extraction of mitten crab Leucodermia virus DNA: the gill getting 50-100mg mitten crab, hepatopancreas, muscle tissue, add the distilled water of 600 μ L sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 10 minutes, get supernatant, add the saturated phenol of 200 μ L Tris-, after abundant vibration mixing, leave standstill 5 minutes, then centrifugal 5 minutes of 12000rpm, get the phenol that supernatant liquor adds equivalent: chloroform: isoamyl alcohol extraction secondary, get the Virahol that supernatant adds 2 times of volumes, after mixing, leave standstill 10 minutes, centrifugal 10 minutes of 12000rpm, after removing supernatant, add the ethanol of 75% of 1mL precooling, leave standstill 5 minutes, centrifugal 10 minutes of 12000rpm, remove supernatant, after being placed in vacuum drying oven drying, with the ddH of 100 μ L
2o dissolves, and be test DNA profiling, the DNA profiling of extraction is placed in-70 DEG C and saves backup,
(2) PCR reaction system: the PCR reaction system adopting 20 μ L, 2 × reaction cocktail buffer 10 μ L is added respectively in 0.2mL PCR reaction tubes, the each 0.5 μ L of upstream primer, downstream primer, 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, DNA profiling 3.0 μ L, adds to cumulative volume with sterilizing ultrapure water, use positive control solution and negative controls as template respectively simultaneously, the positive and negative control group are set, mix the rear centrifugal several seconds, be placed in PCR and react on instrument;
(4) PCR reaction conditions: 95 DEG C of denaturations 5 minutes; Then 95 DEG C of sex change 30 seconds, 56 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, circulate 30 times altogether; 72 DEG C extend 10 minutes, last 4 DEG C of preservations;
(5) after the detection of pcr amplification product: PCR reaction terminates, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR primer, check the expection size of PCR primer;
(6) result and judgement: observe under ultraviolet lamp and contrast with standard molecular weight, if detect sample to occur bright band at 441bp place, and negative control does not have object band, be then that Leucodermia virus is positive; If the visible object band of positive control, and detect sample without object band and occur, then be feminine gender, do not have or be not the object Leucodermia virus that will detect.
Compared with prior art, the specific PCR quick detection kit of mitten crab Leucodermia virus of the present invention and detection method thereof have following characteristics and advantage:
(1) the present invention adopts the method for polymerase chain reaction (polymerase chain reaction, PCR) technology for detection mitten crab Leucodermia virus, is applicable to carry out rapid detection to mitten crab Leucodermia virus.
(2) compared with prior art, beneficial effect of the present invention comprises: 1. detection is quick, efficiency is high: judge only to need 3 hours from nucleic acid extraction, polymerase chain reaction (PCR) to result; Once can carry out the detection of multiple sample, there is high efficiency.2. detect accurately: detect primer and be only combined specifically with mitten crab Leucodermia virus, all there is no cross reaction with other cause of diseases.3. detection sensitivity is high, is suitable for the early stage monitoring of mitten crab Leucodermia virus.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result infecting the Leucodermia virus mitten crab gill, hepatopancreas, muscle tissue.Wherein, in X-coordinate: label 1 is negative control (using negative controls as template); Label 2 is positive control (using positive control solution as template); Label 3 is Leucodermia virus infection Tissue of Eriocheir Sinensis sample; Label M is molecular weight standard DL2000 (purchased from precious biotechnology company limited); In ordinate zou: bp is base pair, 2000,1000,750,500,250,100 is the quantity of the base pair of homologous segment.
Fig. 2 is the pcr amplification result to Different Kinds of Pathogens.Wherein, in X-coordinate: label 1 is Leucodermia virus; Label 2 is spiral shell substance; Label 3 is Aeromonas hydrophila; Label 4 is infectious spleen and kidney necrosis virus; Label 5 is mandarin fish rhabdovirus; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, 2000,1000,750,500,250,100 is the quantity of the base pair of homologous segment.
Fig. 3 gets by same pond the pcr amplification result of the Tissue of Eriocheir Sinensis sample of 4 morbidities.Wherein, in X-coordinate: label 1 is negative control (using negative controls as template); Label 2 is positive control (using positive control solution as template); Label 3-6 is 4 mitten crab tissue samples; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, 2000,1000,750,500,250,100 is the quantity of the base pair of homologous segment.
Fig. 4 gets by same pond the pcr amplification result of the Tissue of Eriocheir Sinensis samples of 2 morbidity Procambius clarkiis and 4 morbidities.Wherein, in X-coordinate: label 1 is negative control (using negative controls as template); Label 2 is positive control (using positive control solution as template); Label 3-4 is 2 Procambius clarkii tissue samples; Label 5-8 is 4 mitten crab tissue samples; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, 2000,1000,750,500,250,100 is the quantity of the base pair of homologous segment.
Fig. 5 gets by different areas the pcr amplification result of 10 mitten crab tissue samples.Wherein, in X-coordinate: label 1 is negative control (using negative controls as template); Label 2 is positive control (using positive control solution as template); Label 3-6 is by being got Tissue of Eriocheir Sinensis sample in 4 Jiangsu Provinces; Label 7-10 is by being got Tissue of Eriocheir Sinensis sample in 4 Anhui provinces; Label 11-12 is by being got Tissue of Eriocheir Sinensis sample in 2 Hubei province; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, 2000,1000,750,500,250,100 is the quantity of the base pair of homologous segment.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail:
Embodiment one: mitten crab Leucodermia virus PCR quick detection kit
All chemical reagent and the primer of the mitten crab Leucodermia virus PCR quick detection kit described in the present embodiment are all bought from the Reagent Company of specialty.But mentioned reagent and Primer Source do not form any limitation of the invention, the present invention can prepare associated reagents and the relevant primer of synthesis voluntarily.
Test kit is made up of (9 sample part) following part:
(1) 2 × reaction cocktail buffer 1.0mL, comprises following composition:
(2) detect primer: upstream primer WSSV-F:5 '-GTGTACTAGGAATATTGGAAT-3 ', downstream primer WSSV-R:5 '-CGGCATTCTTCATGGCTTCTG-3 ', concentration is 10 μMs, and upstream and downstream primer mixes, 400 μ L.
(3) Taq enzyme 5U/ μ L.
(4) ddH of sterilizing
2o 1.0mL.
(5) positive control solution: Leucodermia virus genomic dna.
(6) negative controls: ddH
2o.
(7) process specifications is a.
(8) rectangular parallelepiped box, can load onto and state 1 ~ No. 7 parts, 9.0 × 5.0 × 5.0cm
3.
(9) one pieces of fiber boards, its size is identical with the bottom surface of box, high 2.5cm, has 2 rounds, and often arrange 4 holes, aperture 1.0cm, above-mentioned each tubule respectively correspondence is positioned in these apertures, is loaded in rectangular parallelepiped box.
Above-mentioned positive control is the gill and the hepatic tissue of getting the mitten crab that 100mg Leucodermia virus infects, add the distilled water of 600 μ L sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 10 minutes, get supernatant, add the saturated phenol of 200 μ L Tris-, after abundant vibration mixing, leave standstill 5 minutes, then centrifugal 5 minutes of 12000rpm, get the phenol that supernatant liquor adds equivalent: chloroform: isoamyl alcohol extraction secondary, get the Virahol that supernatant adds 2 times of volumes, after mixing, leave standstill 10 minutes, centrifugal 10 minutes of 12000rpm, after removing supernatant, add the ethanol of 75% of 1mL precooling, leave standstill 5 minutes, centrifugal 10 minutes of 12000rpm, remove supernatant, after being placed in vacuum drying oven drying, with the ddH of 100 μ L
2o dissolves and obtains.
Pcr amplification reaction system (20 μ L) is:
Pcr amplification reaction condition is:
95℃ 5min
1 circulation
95℃ 30s
56℃ 30s
72℃ 40s
30 circulations
72℃ 10min
1 circulation
Amplified production 4 DEG C preservation.
Embodiment two: mitten crab Leucodermia virus PCR detection method
Use the test kit described in embodiment one, follow these steps to carry out:
(1) 50mg measuring samples is got, add the distilled water of 600 μ L sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 10 minutes, get supernatant, add the saturated phenol of 200 μ L Tris-, after abundant vibration mixing, leave standstill 5 minutes, then centrifugal 5 minutes of 12000rpm, get the phenol that supernatant liquor adds equivalent: chloroform: isoamyl alcohol extraction secondary, get the Virahol that supernatant adds 2 times of volumes, after mixing, leave standstill 10 minutes, centrifugal 10 minutes of 12000rpm, after removing supernatant, add the ethanol of 75% of 1mL precooling, leave standstill 5 minutes, centrifugal 10 minutes of 12000rpm, remove supernatant, after being placed in vacuum drying oven drying, with the ddH of 100 μ L
2o dissolves, as the template of PCR reaction.
(2) 2 × reaction cocktail buffer 10 μ L is got respectively, each 0.5 μ L of upstream and downstream primer (WSSV-F, WSSV-R), Taq archaeal dna polymerase 0.5 μ L, ddH
2o 5.5 μ L, template 3.0 μ L, separately gets positive control solution and each 3 μ L of negative controls as template, as positive and negative control group.Mix the rear centrifugal several seconds, be placed in PCR and react on instrument.
(3) pcr amplification is carried out by following condition: 95 DEG C of 5min, 1 circulation; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min, last 4 DEG C of preservations.
(4) after reaction terminates, get 10 μ L and add 2 μ L tetrabromophenol sulfonphthalein mixings, after 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL) electrophoresis 25min, observe on ultraviolet device, if there is bright reaction band at 441bp place, be then that Leucodermia virus is positive; If reactionless band occurs, be then negative.
(5) interpretation: as shown in Figure 1, there is bright reaction band in 441bp place in the electrophoresis band of label 3 (testing sample), also same reaction band is there is in the electrophoresis band of label 2 (positive control), and in the electrophoresis band of label 1 (negative control), there is not reacting band, show that the Detection results of this PCR kit meets expection.
Embodiment three: mitten crab Leucodermia virus PCR quick detection kit specificity experiments
Use the test kit described in embodiment one, follow these steps to carry out:
(1) respectively using the nucleic acid of the Leucodermia virus extracted, spiral shell substance, Aeromonas hydrophila, infectious spleen and kidney necrosis virus, mandarin fish rhabdovirus as template, PCR detection is carried out.
(2) 2 × reaction cocktail buffer 10 μ L is got respectively, each 0.5 μ L of upstream and downstream primer (WSSV-F, WSSV-R), Taq archaeal dna polymerase 0.5 μ L, ddH
2o 5.5 μ L, template 3.0 μ L.Mix the rear centrifugal several seconds, be placed in PCR and react on instrument.
(3) pcr amplification is carried out by following condition: 95 DEG C of 5min, 1 circulation; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min, last 4 DEG C of preservations.
(4) after reaction terminates, get 10 μ L and be added to 2 μ L tetrabromophenol sulfonphthalein mixings, after 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL) electrophoresis 25min, observe on ultraviolet device, if there is bright reaction band at 441bp place, be then that Leucodermia virus is positive; If reactionless band occurs, be then negative.
(5) interpretation: as shown in Figure 2, there is bright reaction band at 441bp place in the electrophoresis band of label 1, display rickettsia-like organism is positive.At 441bp place, reactionless band occurs the electrophoresis band of label 2 ~ 5 (being respectively spiral shell substance, Aeromonas hydrophila, infectious spleen and kidney necrosis virus, mandarin fish rhabdovirus), and display Leucodermia virus is negative.Show the high specificity of test kit of the present invention to Leucodermia virus.
Embodiment four: mitten crab Leucodermia virus PCR quick detection kit is to the detection of morbidity mitten crab
Use the test kit described in embodiment one, follow these steps to carry out:
(1) respectively using from same pond get the DNA that extracts in the mitten crab gills of 4 morbidities and hepatic tissue and, as template, carry out PCR detection.
(2) 2 × reaction cocktail buffer 10 μ L is got respectively, each 0.5 μ L of upstream and downstream primer (WSSV-F, WSSV-R), Taq archaeal dna polymerase 0.5 μ L, ddH
2o 5.5 μ L, template 3.0 μ L, separately gets positive control solution and each 3 μ L of negative controls as template, as positive and negative control group.Mix the rear centrifugal several seconds, be placed in PCR and react on instrument.
(3) pcr amplification is carried out by following condition: 95 DEG C of 5min, 1 circulation; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min, last 4 DEG C of preservations.
(4) after reaction terminates, get 10 μ L and be added to 2 μ L tetrabromophenol sulfonphthalein mixings, after 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL) electrophoresis 25min, observe on ultraviolet device, if there is bright reaction band at 441bp place, be then that Leucodermia virus is positive; If reactionless band occurs, be then negative.
(5) interpretation: detect institute and get in 4 mitten crabs, 4 mitten crab Leucodermia virus are all positive, and show by checking order further, its detected result is Leucodermia virus, and nucleotide homology is all more than 99%, PCR detected result is shown in Fig. 3.
Embodiment five: mitten crab Leucodermia virus PCR quick detection kit is to the detection of fall ill Procambius clarkii and mitten crab
Use the test kit described in embodiment one, follow these steps to carry out:
(1) respectively using from same pond get the DNA that extracts in the mitten crab gills of 2 morbidity Procambius clarkiis and 4 morbidities and hepatic tissue and, as template, carry out PCR detection.
(2) 2 × reaction cocktail buffer 10 μ L is got respectively, each 0.5 μ L of upstream and downstream primer (WSSV-F, WSSV-R), Taq archaeal dna polymerase 0.5 μ L, ddH
2o 5.5 μ L, template 3.0 μ L, separately gets positive control solution and each 3 μ L of negative controls as template, as positive and negative control group.Mix the rear centrifugal several seconds, be placed in PCR and react on instrument.
(3) pcr amplification is carried out by following condition: 95 DEG C of 5min, 1 circulation; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min, last 4 DEG C of preservations.
(4) after reaction terminates, get 10 μ L and be added to 2 μ L tetrabromophenol sulfonphthalein mixings, after 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL) electrophoresis 25min, observe on ultraviolet device, if there is bright reaction band at 441bp place, be then that Leucodermia virus is positive; If reactionless band occurs, be then negative.
(5) interpretation: detect in institute 2 Procambius clarkiis of getting and 4 mitten crabs, 2 Procambius clarkiis and 4 mitten crab Leucodermia virus are all positive, show by checking order further, its detected result is Leucodermia virus, and nucleotide homology is all more than 99%, PCR detected result is shown in Fig. 4.
Embodiment six: mitten crab Leucodermia virus PCR quick detection kit is to the detection of different areas morbidity mitten crab
Use the test kit described in embodiment one, follow these steps to carry out:
(1) respectively using Jiangsu, Anhui, Hubei province get the DNA that extracts in 10 mitten crab gills and hepatic tissue as template, carry out PCR detection.
(2) 2 × reaction cocktail buffer 10 μ L is got respectively, each 0.5 μ L of upstream and downstream primer (WSSV-F, WSSV-R), Taq archaeal dna polymerase 0.5 μ L, ddH
2o 5.5 μ L, template 3.0 μ L, separately gets positive control solution and each 3 μ L of negative controls as template, as positive and negative control group.Mix the rear centrifugal several seconds, be placed in PCR and react on instrument.
(3) pcr amplification is carried out by following condition: 95 DEG C of 5min, 1 circulation; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min, last 4 DEG C of preservations.
(4) after reaction terminates, get 10 μ L and be added to 2 μ L tetrabromophenol sulfonphthalein mixings, after 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL) electrophoresis 25min, observe on ultraviolet device, if there is bright reaction band at 441bp place, be then that Leucodermia virus is positive; If reactionless band occurs, be then negative.
(5) interpretation: detect institute and get in 10 mitten crabs, 10 mitten crab Leucodermia virus are all positive, and show by checking order further, its detected result is Leucodermia virus, and nucleotide homology is all more than 99%, PCR detected result is shown in Fig. 5.
Claims (2)
1. the PCR quick detection kit of mitten crab Leucodermia virus, is characterized in that, described test kit comprises:
(1) 2 × reaction cocktail buffer, comprises following composition: KCl, MgCl
2, Tris-HCl pH5.8, dNTP;
(2) design and synthesis of primer is detected: according to the conserved sequence of the Leucodermia virus gene issued in GenBank, design a pair specific detection primer, upstream primer WSSV-F:5 '-GTGTACTAGGAATATTGGAAT-3 ', downstream primer WSSV-R:5 '-CGGCATTCTTCATGGCTTCTG-3 ';
(3) Taq enzyme;
(4) ddH of sterilizing
2o;
(5) positive control solution: Leucodermia virus genomic dna;
(6) negative controls: ddH
2o.
2. test kit according to claim 1, is characterized in that, the method adopting test kit to detect mitten crab Leucodermia virus is:
(1) extraction of mitten crab Leucodermia virus DNA: the gill getting mitten crab, hepatopancreas, muscle tissue, add the distilled water of sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 10 minutes, get supernatant, add the saturated phenol of Tris-, after abundant vibration mixing, leave standstill 5 minutes, then centrifugal 5 minutes of 12000rpm, get the phenol that supernatant liquor adds equivalent: chloroform: isoamyl alcohol extraction secondary, get the Virahol that supernatant adds 2 times of volumes, after mixing, leave standstill 10 minutes, centrifugal 10 minutes of 12000rpm, after removing supernatant, add the ethanol of 75%, leave standstill 5 minutes, centrifugal 10 minutes of 12000rpm, remove supernatant, after being placed in vacuum drying oven drying, use ddH
2o dissolves, and be test DNA profiling, the DNA profiling of extraction is placed in-70 DEG C and saves backup,
(2) PCR reaction system: adopt PCR reaction system, 2 × reaction cocktail buffer is added respectively in PCR reaction tubes, upstream primer, downstream primer, 5U/ μ L Taq archaeal dna polymerase, DNA profiling, adds sterilizing ultrapure water, use positive control solution and negative controls as template respectively simultaneously, the positive and negative control group are set, mix the rear centrifugal several seconds, be placed in PCR and react on instrument;
(4) PCR reaction conditions: 95 DEG C of denaturations 5 minutes; Then 95 DEG C of sex change 30 seconds, 56 DEG C of annealing 30 seconds, 72 DEG C extend 40 seconds, circulate 30 times altogether; 72 DEG C extend 10 minutes, last 4 DEG C of preservations;
(5) after the detection of pcr amplification product: PCR reaction terminates, get product on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR primer, check the expection size of PCR primer;
(6) result and judgement: observe under ultraviolet lamp and contrast with standard molecular weight, if detect sample to occur bright band at 441bp place, and negative control does not have object band, be then that Leucodermia virus is positive; If the visible object band of positive control, and detect sample without object band and occur, then be feminine gender, do not have or be not the object Leucodermia virus that will detect.
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| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN111850137A (en) * | 2020-07-29 | 2020-10-30 | 扬州福岁乐生物科技有限公司 | Direct PCR method for eriocheir sinensis hemolymph |
| CN114107528A (en) * | 2021-12-10 | 2022-03-01 | 广州双螺旋基因技术有限公司 | Constant-temperature rapid detection kit for detecting eriocheir sinensis spiroplasma |
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| CN110592288A (en) * | 2019-10-31 | 2019-12-20 | 盐城市大丰区水产技术推广站 | RT-PCR detection method for eriocheir sinensis bicistronic virus |
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| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN111850137A (en) * | 2020-07-29 | 2020-10-30 | 扬州福岁乐生物科技有限公司 | Direct PCR method for eriocheir sinensis hemolymph |
| CN114107528A (en) * | 2021-12-10 | 2022-03-01 | 广州双螺旋基因技术有限公司 | Constant-temperature rapid detection kit for detecting eriocheir sinensis spiroplasma |
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